The beneficial use of lipolytic enzymes (E.C. 3.1.1.x) in food and/or feed industrial applications has been known for many years.
For instance, in EP 0 585 988 it is claimed that lipase addition to dough resulted in an improvement in the antistaling effect. It is suggested that a lipase obtained from Rhizopus arrhizus when added to dough can improve the quality of the resultant bread when used in combination with shortening/fat. WO94/04035 teaches that an improved bread softness can be obtained by adding a lipase to dough without the addition of any additional fat/oil to the dough. Castello, P. ESEGP 89-10 December 1999 Helsinki, shows that exogenous lipases can modify bread volume.
The substrate for lipases in wheat flour is 1.5-3% endogenous wheat lipids, which are a complex mixture of polar and non-polar lipids. The polar lipids can be divided into glycolipids and phospholipids. These lipids are built up of glycerol esterified with two fatty acids and a polar group. The polar group contributes to surface activity of these lipids. Enzymatic cleavage of one of the fatty acids in these lipids leads to lipids with a much higher surface activity. It is well known that emulsifiers, such as DATEM, with high surface activity are very functional when added to dough.
Lipolytic enzymes hydrolyse one or more of the fatty acids from lipids present in the food which can result in the formation of powerful emulsifier molecules within the foodstuff which provide commercially valuable functionality. The molecules which contribute the most significant emulsifier characteristics are the partial hydrolysis products, such as lyso-phospholipids, lyso-glycolipids and mono-glyceride molecules. The polar lipid hydrolysis products, namely lyso-phospholipids and lyso-glycolipids, are particularly advantageous. In bread making, such in situ derived emulsifiers can give equivalent functionality as added emulsifiers, such as DATEM.
However, the activity of lipolytic enzymes has also been found to result in accumulation of free fatty acids, which can lead to detrimental functionality in the foodstuff. This inherent activity of lipolytic enzymes limits their functionality.
The negative effect on bread volume is often explained by overdosing. Overdosing can lead to a decrease in gluten elasticity which results in a dough which is too stiff and thus results in reduced volumes. In addition, or alternatively, such lipases can degrade shortening, oil or milk fat added to the dough, resulting in off-flavour in the dough and baked product. Overdosing and off-flavour have been attributed to the accumulation of free fatty acids in the dough, particularly short chain fatty acids.
The presence of high levels of free fatty acids (FFA) in raw materials or food products is generally recognised as a quality defect and food processors and customers will usually include a maximum FFA level in the food specifications. The resulting effects of excess FFA levels can be in organoleptic and/or functional defects.
In EP 1 193 314, the inventors discovered that the use of lipolytic enzymes active on glycolipids was particularly beneficial in applications in bread making, as the partial hydrolysis products the lyso-glycolipids were found to have very high emulsifier functionality, apparently resulting in a higher proportion of positive emulsifier functionality compared to the detrimental accumulation of free fatty acids. However, the enzymes were also found to have significant non-selective activity on triglycerides which resulted in unnecessarily high free fatty acid.
This problem of high triglyceride activity was addressed in WO 02/094123, where the inventors discovered that by selecting lipolytic enzymes which were active on the polar lipids (glycolipids and phospholipids) in a dough, but substantially not active on triglycerides or 1-mono-glycerides, an improved functionality could be achieved.
A commercially preferred source of lipase enzymes is filamentous fungi, such as Aspergillus spp. and Fusarium spp. Lipases isolated from filamentous fungi have been found to have industrially applicable characteristics and also have been found to be routine to express in heterologous production systems, such as in Aspergillus oryzae, Fusarium and yeast.
A lipase from Fusarium oxysporum was identified in EP 0 130 064, and the application of F. oxysporum lipases in food applications has been suggested in Hoshino et al. (1992) Biosci. Biotech. Biochem 56: 660-664.
EP0 869 167 describes the cloning and expression of a Fusarium oxysporum lipase and its use in baking. The enzyme is described as having phospholipase activity. This enzyme is now sold by Novozymes A/S (Denmark) as Lipopan F™.
WO 02/00852 discloses five lipase enzymes and their encoding polynucleotides, isolated from F. venenatum, F. sulphureum, A. berkeleyanum, F. culmorum and F. solani. All five enzymes are described as having triacylglycerol hydrolysing activity, phospholipase and galactolipase activity. Three of the enzymes have equivalent activity to the F. oxysporum enzyme taught in EP 0 869 167: F. venenatum, F. sulphureum, F. culmorum. 
Therefore, it is apparent that some Fusarium lipases, including Lipopan F™ have been found to have side activity on polar lipids, including phospholipids and glycolipids. Although described as a phospholipase in EP 0 869 167, the lipase from Fusarium oxysporum has high lipase activity. The enzyme also has glycolipase activity. However, despite the significant activity on polar lipids, the functionality achieved by use of the enzyme is limited due to the high lipase (i.e. triglyceride) activity.
Nagao et al (J. Biochem 116 (1994) 536-540) describes a lipase from F. heterosporum; which enzyme predominantly functions as a lipase (E.C. 3.1.1.3) to hydrolyse triglycerides. This is very different from the enzymes according to the present invention.
Lipolytic enzyme variants, with specific amino acid substitutions and fusions, have been produced some of which have an enhanced activity on the polar lipids compared to the wild-type parent enzymes. WO01/39602 describes such a variant, referred to as SP979, which is a fusion of the Thermomyces lanuginosus lipase, and the Fusarium oxysporum lipase described in EP 0 869 167. This variant has been found to have a significantly high ratio of activity on phospholipids and glycolipids compared to triglycerides.
However, prior to the present invention, natural fungal lipolytic enzymes, particularly from Fusarium spp., having a high ratio of activity on polar lipids compared with triglycerides had not been taught.